Chitinase-h (Chi-h) is of special interest among insect chitinases due to its exclusive distribution in lepidopteran insects and high sequence identity with bacterial and baculovirus homologs. Here OfChi-h, a Chi-h from Ostrinia furnacalis, was investigated. Crystal structures of both OfChi-h and its complex with chitoheptaose ((GlcN)7) reveal that OfChi-h possesses a long and asymmetric substrate-binding cleft, which is a typical characteristics of a processive exo-chitinase. The structural comparison between OfChi-h and its bacterial homolog SmChiA uncovered two phenylalanine to tryptophan site variants in OfChi-h at subsites +2 and possibly -7. The F232W/F396W double mutant endowed SmChiA with higher hydrolytic activities toward insoluble substrates, such as insect cuticle, α-chitin and chitin nanowhisker. Enzymatic assay demonstrated that OfChi-h outperformed OfChtI, an insect endo-chitinase, toward the insoluble substrates, but showed lower activity toward the soluble substrate ethylene glycol chitin. Furthermore, OfChi-h was found to be inhibited by N,N′,N′′-trimethylglucosamine-N,N′,N′′,N′′′-tetraacetylchitotetraose (TMG-(GlcNAc)4), a substrate analog which can be degraded into TMG-(GlcNAc)1-2. Injection of TMG-(GlcNAc)4 into 5th instar O. furnacalis larvae led to severe defects in pupation. This work provides insights into a molting-indispensable insect chitinase that is phylogenetically closer to bacterial chitinases than insect chitinases.
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